We report the raw signal data output obtained from a <em>Malassezia pachydermatis</em>... more We report the raw signal data output obtained from a <em>Malassezia pachydermatis</em> isolate of a canine clinical sample (otitis). Multiple barcoded samples run was performed with the MinION device, Oxford Nanopore Technologies, and the barcode02 was assigned to this sample. The raw signal data, fast5 files (showed in the Fats5.zip directory), were basecalled and demultiplexed in order to achieve the fastq file format which includes only the sequences targeted with barcode02 (M13_UAB2019.fastq).
Journal of Antimicrobial Chemotherapy, Nov 17, 2020
To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia c... more To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates between different healthcare institutions in Catalonia, Spain. Methods: Antimicrobial susceptibility was tested by disc diffusion. MICs were determined by gradient diffusion or broth microdilution. Carbapenemase production was confirmed by lateral flow. PCR and Sanger sequencing were used to identify the allelic variants of resistance genes. Clonality studies were performed by PFGE and MLST. Plasmid typing, conjugation assays, S1-PFGE plus Southern blotting and MinION Oxford Nanopore sequencing were used to characterize resistance plasmids. Results: Twenty-nine carbapenem-resistant isolates recovered from three healthcare institutions between January and November 2016 were included: 14 K. pneumoniae isolates from a tertiary hospital in the south of Catalonia (hospital A); 2 K. pneumoniae isolates from a nearby healthcare centre; and 12 K. pneumoniae isolates and 1 E. coli isolate from a tertiary hospital in Barcelona (hospital B). The majority of isolates were resistant to all antimicrobial agents, except colistin, and all were NDM producers. PFGE identified a major K. pneumoniae clone (n = 27) belonging to ST147 and co-producing NDM-1 and CTX-M-15, with a few isolates also harbouring bla OXA-48. Two sporadic isolates of K. pneumoniae ST307 and E. coli ST167 producing NDM-7 were also identified. bla NDM-1 was carried in two related IncR plasmid populations and bla NDM-7 in a conjugative 50 kb IncX3 plasmid. Conclusions: We report the inter-hospital dissemination of XDR high-risk clones of K. pneumoniae and E. coli associated with the carriage of small, transferable plasmids harbouring bla NDM genes.
ABSTRACT A Nomenclature Committee for factors of the dog major histocompatibility system or dog l... more ABSTRACT A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence-based nomenclature for the genes of the DLA system. The remit of this committee includes: i) assignment of gene names; ii) rules for naming alleles; iii) assignment of names to published alleles; iv) assignment of names to new alleles; and v) rules for acceptance of new alleles.
Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del ... more Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del Cadí (foto: Gabriel Lampreave).
The presence of Bartonella spp. was detected by polymerase chain reaction (PCR) in dogs from Spai... more The presence of Bartonella spp. was detected by polymerase chain reaction (PCR) in dogs from Spain with blood culture-negative endocarditis. The aim of this study is to add information about canine infectious endocarditis in Europe. Animals: Thirty dogs with naturally occurring blood culture-negative endocarditis were examined from 2010 to 2017 at three veterinary referral hospitals, located in northwest, northeast, and southeast of Spain.
Background: Modern dog breeds display traits that are either breed-specific or shared by a few br... more Background: Modern dog breeds display traits that are either breed-specific or shared by a few breeds as a result of genetic bottlenecks during the breed creation process and artificial selection for breed standards. Selective sweeps in the genome result from strong selection and can be detected as a reduction or elimination of polymorphism in a given region of the genome. Results: Extended regions of homozygosity, indicative of selective sweeps, were identified in a genome-wide scan dataset of 25 Boxers from the United Kingdom genotyped at~20,000 single-nucleotide polymorphisms (SNPs). These regions were further examined in a second dataset of Boxers collected from a different geographical location and genotyped using higher density SNP arrays (~170,000 SNPs). A selective sweep previously associated with canine brachycephaly was detected on chromosome 1. A novel selective sweep of over 8 Mb was observed on chromosome 26 in Boxer and for a shorter region in English and French bulldogs. It was absent in 171 samples from eight other dog breeds and 7 Iberian wolf samples. A region of extended increased heterozygosity on chromosome 9 overlapped with a previously reported copy number variant (CNV) which was polymorphic in multiple dog breeds. Conclusion: A selective sweep of more than 8 Mb on chromosome 26 was identified in the Boxer genome. This sweep is likely caused by strong artificial selection for a trait of interest and could have inadvertently led to undesired health implications for this breed. Furthermore, we provide supporting evidence for two previously described regions: a selective sweep on chromosome 1 associated with canine brachycephaly and a CNV on chromosome 9 polymorphic in multiple dog breeds.
bioRxiv (Cold Spring Harbor Laboratory), Feb 6, 2020
The availability of long-read technologies, like Oxford Nanopore Technologies, provides the oppor... more The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities in real-time and up to the species level. We assess amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What's in my pot (WIMP) classifier. We used the ZymoBIOMICS TM mock community and different fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community, as well as Malassezia pachydermatis, Microsporum canis, Aspergillus fumigatus from the microbiological cultures, and identified Rhodotorula graminis as the species mislabelled as Candida spp in the previous culture. We applied the same approach to communities, such as external otitis in dogs. Malassezia spp was found as the dominant fungi in the ear skin. We identified M. pachydermatis as the main species in the healthy sample, and a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of this approach to characterize the fungal community of complex samples, either healthy samples or samples with clinical signs of infection. .
Profiling the microbiome of low-biomass samples is Background: challenging for metagenomics since... more Profiling the microbiome of low-biomass samples is Background: challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). We sequenced a clinical isolate of Methods: Staphylococcus , two mock communities and two pools of low-biomass pseudintermedius samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with database. Consensus rrn sequences of the 16S-ITS-23S genetic marker were obtained using canu. The full-length 16S rRNA and the 16S-ITS-23S region of the Results: rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species iñ 98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Both full-length 16S rRNA and the 16S-ITS-23S of the Conclusions: rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the
A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte ... more A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence-based nomenclature for the genes of the DLA system. The remit of this committee includes: i) assignment of gene names; ii) rules for naming alleles; iii) assignment of names to published alleles; iv) assignment of names to new alleles; and v) rules for acceptance of new alleles. This first International Society for Animal Genetics (ISAG) dog major histocompatibility system (MHC) Nomenclature Report considers the rules for acceptance of dog leukocyte antigen (DLA) genes and alleles, together with an appropriate sequence based nomenclature. Names have been assigned to existing sequences, where appropriate. The report also includes sequence alignments (both nucleotide and amino acid) for DLA class II alleles, including data which has not been previously published. There is a table for each class II locus, which lists all published and unpublished sequence data, with accession numbers and references, plus equivalents for new and previously used allele names. The Committee would like to acknowledge the contribution of the IUIS Committee on Nomenclature of DLA Determinants (1) (a subcommittee of the Nomenclature Committee of the International Union of Immunological Societies), which started this work over 20 years ago. In 1990, Klein (2) proposed the use of the term Cafa (from Canis familiaris) to describe the class I and II genes in the dog MHC region. However, many publications before and since have continued to use the term DLA. The Nomenclature Committee has affirmed the use of the term DLA. This proposal has the approval of Dr. Ronald Bontrop, the Editor of Immunogenetics, who maintains the register of MHC symbols for all species.
The International Society for Animal Genetics (ISAG) Dog Leukocyte Antigen (DLA) Nomenclature Com... more The International Society for Animal Genetics (ISAG) Dog Leukocyte Antigen (DLA) Nomenclature Committee met during the ÔComparative Evolution of the Mammalian major Histocompatibility Complex (MHC)Õ meeting in Manchester, UK on 10 September 2000. The main points discussed were the naming of class I genes and alleles, and the inclusion of alleles from other canidae.
Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del ... more Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del Cadí (foto: Gabriel Lampreave).
Background. Studies using highly sensitive molecular techniques have detected bacterial communiti... more Background. Studies using highly sensitive molecular techniques have detected bacterial communities below the human epidermis. Depending on their abundance and composition, this nding could be clinically relevant. This possibility, however, has not been investigated in the dog so far. The aim of this study was to determine if bacteria can be detected in the dermis and subcutaneous tissue of healthy dogs using two different approaches: traditional cultures and DNA sequencing of the V4 region of bacterial 16S rRNA gene using next-generation sequencing (NGS). Results. Seven healthy dogs were included in the study, and two sets of samples were collected from each subject. Sample sets were composed of a 6-mm abdominal skin biopsy, including epidermis, dermis, and subcutis, a skin surface swab, and an environmental blank sample for contamination control. One set of samples from each dog was submitted for bacterial culture and the other one for bacterial DNA ampli cation and sequencing. Five different bacterial genera (Staphylococcus, Bacillus, Corynebacterium, Streptococcus, and Enterococcus) were isolated in ve out of the seven skin surface swab samples with aerobic microbiological culture methods, while no growth was obtained from the other two samples. Although some DNA could be ampli ed from epidermal, dermal, and subcutaneous tissue samples, the results of the NGS were similar to those of the blanks. Conclusion. When investigated with aerobic microbiological culture methods, the dermis and subcutaneous tissue of dogs are sterile. NGS techniques lead to the detection of some bacterial DNA, similar to the signal detected in blanks, which does not support the presence of a microbiota in dermis or subcutaneous tissue.
The human gut microbiome has been extensively studied, yet the canine gut microbiome is still lar... more The human gut microbiome has been extensively studied, yet the canine gut microbiome is still largely unknown. The availability of high-quality genomes is essential in the fields of veterinary medicine and nutrition to unravel the biological role of key microbial members in the canine gut environment. Our aim was to evaluate nanopore long-read metagenomics and Hi-C (high-throughput chromosome conformation capture) proximity ligation to provide high-quality metagenome-assembled genomes (HQ MAGs) of the canine gut environment. By combining nanopore long-read metagenomics and Hi-C proximity ligation, we retrieved 27 HQ MAGs and 7 medium-quality MAGs of a faecal sample of a healthy dog. Canine MAGs (CanMAGs) improved genome contiguity of representatives from the animal and human MAG catalogues – short-read MAGs from public datasets – for the species they represented: they were more contiguous with complete ribosomal operons and at least 18 canonical tRNAs. Both canine-specific bacterial species and gut generalists inhabit the dog’s gastrointestinal environment. Most of them belonged to Firmicutes , followed by Bacteroidota and Proteobacteria . We also assembled one Actinobacteriota and one Fusobacteriota MAG. CanMAGs harboured antimicrobial-resistance genes (ARGs) and prophages and were linked to plasmids. ARGs conferring resistance to tetracycline were most predominant within CanMAGs, followed by lincosamide and macrolide ones. At the functional level, carbohydrate transport and metabolism was the most variable within the CanMAGs, and mobilome function was abundant in some MAGs. Specifically, we assigned the mobilome functions and the associated mobile genetic elements to the bacterial host. The CanMAGs harboured 50 bacteriophages, providing novel bacterial-host information for eight viral clusters, and Hi-C proximity ligation data linked the six potential plasmids to their bacterial host. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAG discovery and assignment of extra-chromosomal elements to their bacterial host. This will provide essential information for studying the canine gut microbiome in veterinary medicine and animal nutrition.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 M... more We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 Mbp size and 99.43% completeness, 2,386 coding sequences, 19 complete rRNAs, 59 tRNAs, and 4 noncoding RNAs. We released 51 single-contig complete genomes and 16 genomes with a circular main contig using Nanopore sequencing. S taphylococcus pseudintermedius is a commensal of the skin of dogs and the main causative agent of canine pyoderma (1), but populations inhabiting the skin of healthy dogs are largely unknown. The current paradigm indicates that infection arises when the skin barrier is altered by predisposing factors (2). We previously demonstrated that Nanopore sequencing allows de novo assembly of the entire genome of S. pseudintermedius (3). Here, we retrieved 67 S. pseudintermedius genomes from isolates from the skin of healthy dogs using long-read Nanopore sequencing. Samples were obtained by rubbing sterile swabs on four skin sites from nine healthy dogs-perinasal, perioral, inguinal, and perianal. After culture in blood agar at 37°C for 24 h, colonies grown with the morphology of S. pseudintermedius (small silver colonies) were seeded in 3 mL of brain heart infusion (BHI) at 37°C for 16 h. DNA was extracted with a ZymoBIOMICS DNA miniprep kit (Zymo Research). DNA quality and quantity were determined using a NanoDrop 2000 instrument and a Qubit double-stranded DNA (dsDNA) broad range (BR) assay kit (Fisher Scientific). The sequencing libraries were prepared with the rapid barcoding sequencing kit (SQK-RBK004; Oxford Nanopore Technologies [ONT]). Up to 12 barcoded samples were loaded in a MinION FLO-MIN106 9.4.1 flow cell and sequenced in a MinION Mk1B or Mk1C device (ONT). The fast5 files were basecalled and demultiplexed, and adapters were trimmed with Guppy 5.0.11 (4) (ONT) (-dna_r9.4.1_450bps_sup.cfg) (-config configuration.cfg-barcode_kits SQK-RBK004-trim_barcodes; min_score threshold default 60). Reads with a quality score lower than 10 were discarded. Run summary statistics were obtained with Nanoplot 1.38.1 (5) (-N50-fastq). Isolates were confirmed as S. pseudintermedius by EPI2ME WIMP workflow (6). Genomes were de novo assembled using Flye 2.8.3 (7) (-nano-raw-plasmids-trestle), except HSP279 and HSP281, which were assembled with Flye 2.9 (-nano-hq). Contigs were polished with medaka 1.4.3 (8) (medaka_consensus;-m r941_min_sup_g507). Genome completeness and contamination were assessed with CheckM 1.1.3 (lineage_wf) (9). Circlator 1.5.5 was used to rotate the genomes, fixing the start with the dnaA gene (10) (fixstart-min_id 70). Genomes
Studies on tick microbial communities historically focused on tick-borne pathogens. However, ther... more Studies on tick microbial communities historically focused on tick-borne pathogens. However, there is an increasing interest in capturing relationships among non-pathogenic endosymbionts and exploring their relevance for tick biology. The present study included a total of 1600 adult ticks collected from domestic dogs in 4 different biogeographical regions of Spain. Each pool formed by 1 to 10 halves of individuals representing one specific ticks species was examined by PCR for the presence of Coxiellaceae, Rickettsia spp., Rickettsiales, Wolbachia spp., and other bacterial DNA. Of the pools analyzed, 92% tested positive for endosymbiont-derived DNA. Coxiella spp. endosymbionts were the most prevalent microorganisms, being always present in Rhipicephalus sanguineus sensu lato (s.l.) pools. Rickettsia spp. DNA was detected in 60% of Dermacentor reticulatus pools and 40% of R. sanguineus s.l. pools, with a higher diversity of Rickettsia species in R. sanguineus s.l. pools. Our study reveals a negative relationship of Rickettsia massiliae with the presence of tick-borne pathogens in the same pool of ticks. An additional endosymbiont, 'Candidatus Rickettsiella isopodorum', was only detected in D. reticulatus pools. Data from this study indicate that dogs in Spain are exposed to several endosymbionts. Due to the importance of tick-borne pathogens, characterizing the role of endosymbionts for tick physiology and prevalence, may lead to novel control strategies.
We report the raw signal data output obtained from a <em>Malassezia pachydermatis</em>... more We report the raw signal data output obtained from a <em>Malassezia pachydermatis</em> isolate of a canine clinical sample (otitis). Multiple barcoded samples run was performed with the MinION device, Oxford Nanopore Technologies, and the barcode02 was assigned to this sample. The raw signal data, fast5 files (showed in the Fats5.zip directory), were basecalled and demultiplexed in order to achieve the fastq file format which includes only the sequences targeted with barcode02 (M13_UAB2019.fastq).
Journal of Antimicrobial Chemotherapy, Nov 17, 2020
To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia c... more To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates between different healthcare institutions in Catalonia, Spain. Methods: Antimicrobial susceptibility was tested by disc diffusion. MICs were determined by gradient diffusion or broth microdilution. Carbapenemase production was confirmed by lateral flow. PCR and Sanger sequencing were used to identify the allelic variants of resistance genes. Clonality studies were performed by PFGE and MLST. Plasmid typing, conjugation assays, S1-PFGE plus Southern blotting and MinION Oxford Nanopore sequencing were used to characterize resistance plasmids. Results: Twenty-nine carbapenem-resistant isolates recovered from three healthcare institutions between January and November 2016 were included: 14 K. pneumoniae isolates from a tertiary hospital in the south of Catalonia (hospital A); 2 K. pneumoniae isolates from a nearby healthcare centre; and 12 K. pneumoniae isolates and 1 E. coli isolate from a tertiary hospital in Barcelona (hospital B). The majority of isolates were resistant to all antimicrobial agents, except colistin, and all were NDM producers. PFGE identified a major K. pneumoniae clone (n = 27) belonging to ST147 and co-producing NDM-1 and CTX-M-15, with a few isolates also harbouring bla OXA-48. Two sporadic isolates of K. pneumoniae ST307 and E. coli ST167 producing NDM-7 were also identified. bla NDM-1 was carried in two related IncR plasmid populations and bla NDM-7 in a conjugative 50 kb IncX3 plasmid. Conclusions: We report the inter-hospital dissemination of XDR high-risk clones of K. pneumoniae and E. coli associated with the carriage of small, transferable plasmids harbouring bla NDM genes.
ABSTRACT A Nomenclature Committee for factors of the dog major histocompatibility system or dog l... more ABSTRACT A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence-based nomenclature for the genes of the DLA system. The remit of this committee includes: i) assignment of gene names; ii) rules for naming alleles; iii) assignment of names to published alleles; iv) assignment of names to new alleles; and v) rules for acceptance of new alleles.
Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del ... more Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del Cadí (foto: Gabriel Lampreave).
The presence of Bartonella spp. was detected by polymerase chain reaction (PCR) in dogs from Spai... more The presence of Bartonella spp. was detected by polymerase chain reaction (PCR) in dogs from Spain with blood culture-negative endocarditis. The aim of this study is to add information about canine infectious endocarditis in Europe. Animals: Thirty dogs with naturally occurring blood culture-negative endocarditis were examined from 2010 to 2017 at three veterinary referral hospitals, located in northwest, northeast, and southeast of Spain.
Background: Modern dog breeds display traits that are either breed-specific or shared by a few br... more Background: Modern dog breeds display traits that are either breed-specific or shared by a few breeds as a result of genetic bottlenecks during the breed creation process and artificial selection for breed standards. Selective sweeps in the genome result from strong selection and can be detected as a reduction or elimination of polymorphism in a given region of the genome. Results: Extended regions of homozygosity, indicative of selective sweeps, were identified in a genome-wide scan dataset of 25 Boxers from the United Kingdom genotyped at~20,000 single-nucleotide polymorphisms (SNPs). These regions were further examined in a second dataset of Boxers collected from a different geographical location and genotyped using higher density SNP arrays (~170,000 SNPs). A selective sweep previously associated with canine brachycephaly was detected on chromosome 1. A novel selective sweep of over 8 Mb was observed on chromosome 26 in Boxer and for a shorter region in English and French bulldogs. It was absent in 171 samples from eight other dog breeds and 7 Iberian wolf samples. A region of extended increased heterozygosity on chromosome 9 overlapped with a previously reported copy number variant (CNV) which was polymorphic in multiple dog breeds. Conclusion: A selective sweep of more than 8 Mb on chromosome 26 was identified in the Boxer genome. This sweep is likely caused by strong artificial selection for a trait of interest and could have inadvertently led to undesired health implications for this breed. Furthermore, we provide supporting evidence for two previously described regions: a selective sweep on chromosome 1 associated with canine brachycephaly and a CNV on chromosome 9 polymorphic in multiple dog breeds.
bioRxiv (Cold Spring Harbor Laboratory), Feb 6, 2020
The availability of long-read technologies, like Oxford Nanopore Technologies, provides the oppor... more The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities in real-time and up to the species level. We assess amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What's in my pot (WIMP) classifier. We used the ZymoBIOMICS TM mock community and different fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community, as well as Malassezia pachydermatis, Microsporum canis, Aspergillus fumigatus from the microbiological cultures, and identified Rhodotorula graminis as the species mislabelled as Candida spp in the previous culture. We applied the same approach to communities, such as external otitis in dogs. Malassezia spp was found as the dominant fungi in the ear skin. We identified M. pachydermatis as the main species in the healthy sample, and a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of this approach to characterize the fungal community of complex samples, either healthy samples or samples with clinical signs of infection. .
Profiling the microbiome of low-biomass samples is Background: challenging for metagenomics since... more Profiling the microbiome of low-biomass samples is Background: challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). We sequenced a clinical isolate of Methods: Staphylococcus , two mock communities and two pools of low-biomass pseudintermedius samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with database. Consensus rrn sequences of the 16S-ITS-23S genetic marker were obtained using canu. The full-length 16S rRNA and the 16S-ITS-23S region of the Results: rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species iñ 98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Both full-length 16S rRNA and the 16S-ITS-23S of the Conclusions: rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the
A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte ... more A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence-based nomenclature for the genes of the DLA system. The remit of this committee includes: i) assignment of gene names; ii) rules for naming alleles; iii) assignment of names to published alleles; iv) assignment of names to new alleles; and v) rules for acceptance of new alleles. This first International Society for Animal Genetics (ISAG) dog major histocompatibility system (MHC) Nomenclature Report considers the rules for acceptance of dog leukocyte antigen (DLA) genes and alleles, together with an appropriate sequence based nomenclature. Names have been assigned to existing sequences, where appropriate. The report also includes sequence alignments (both nucleotide and amino acid) for DLA class II alleles, including data which has not been previously published. There is a table for each class II locus, which lists all published and unpublished sequence data, with accession numbers and references, plus equivalents for new and previously used allele names. The Committee would like to acknowledge the contribution of the IUIS Committee on Nomenclature of DLA Determinants (1) (a subcommittee of the Nomenclature Committee of the International Union of Immunological Societies), which started this work over 20 years ago. In 1990, Klein (2) proposed the use of the term Cafa (from Canis familiaris) to describe the class I and II genes in the dog MHC region. However, many publications before and since have continued to use the term DLA. The Nomenclature Committee has affirmed the use of the term DLA. This proposal has the approval of Dr. Ronald Bontrop, the Editor of Immunogenetics, who maintains the register of MHC symbols for all species.
The International Society for Animal Genetics (ISAG) Dog Leukocyte Antigen (DLA) Nomenclature Com... more The International Society for Animal Genetics (ISAG) Dog Leukocyte Antigen (DLA) Nomenclature Committee met during the ÔComparative Evolution of the Mammalian major Histocompatibility Complex (MHC)Õ meeting in Manchester, UK on 10 September 2000. The main points discussed were the naming of class I genes and alleles, and the inclusion of alleles from other canidae.
Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del ... more Zona frecuentada por el lobo en Cataluña (foto: Jordi Ruiz-Olmo). Lobo avistado en la sierra del Cadí (foto: Gabriel Lampreave).
Background. Studies using highly sensitive molecular techniques have detected bacterial communiti... more Background. Studies using highly sensitive molecular techniques have detected bacterial communities below the human epidermis. Depending on their abundance and composition, this nding could be clinically relevant. This possibility, however, has not been investigated in the dog so far. The aim of this study was to determine if bacteria can be detected in the dermis and subcutaneous tissue of healthy dogs using two different approaches: traditional cultures and DNA sequencing of the V4 region of bacterial 16S rRNA gene using next-generation sequencing (NGS). Results. Seven healthy dogs were included in the study, and two sets of samples were collected from each subject. Sample sets were composed of a 6-mm abdominal skin biopsy, including epidermis, dermis, and subcutis, a skin surface swab, and an environmental blank sample for contamination control. One set of samples from each dog was submitted for bacterial culture and the other one for bacterial DNA ampli cation and sequencing. Five different bacterial genera (Staphylococcus, Bacillus, Corynebacterium, Streptococcus, and Enterococcus) were isolated in ve out of the seven skin surface swab samples with aerobic microbiological culture methods, while no growth was obtained from the other two samples. Although some DNA could be ampli ed from epidermal, dermal, and subcutaneous tissue samples, the results of the NGS were similar to those of the blanks. Conclusion. When investigated with aerobic microbiological culture methods, the dermis and subcutaneous tissue of dogs are sterile. NGS techniques lead to the detection of some bacterial DNA, similar to the signal detected in blanks, which does not support the presence of a microbiota in dermis or subcutaneous tissue.
The human gut microbiome has been extensively studied, yet the canine gut microbiome is still lar... more The human gut microbiome has been extensively studied, yet the canine gut microbiome is still largely unknown. The availability of high-quality genomes is essential in the fields of veterinary medicine and nutrition to unravel the biological role of key microbial members in the canine gut environment. Our aim was to evaluate nanopore long-read metagenomics and Hi-C (high-throughput chromosome conformation capture) proximity ligation to provide high-quality metagenome-assembled genomes (HQ MAGs) of the canine gut environment. By combining nanopore long-read metagenomics and Hi-C proximity ligation, we retrieved 27 HQ MAGs and 7 medium-quality MAGs of a faecal sample of a healthy dog. Canine MAGs (CanMAGs) improved genome contiguity of representatives from the animal and human MAG catalogues – short-read MAGs from public datasets – for the species they represented: they were more contiguous with complete ribosomal operons and at least 18 canonical tRNAs. Both canine-specific bacterial species and gut generalists inhabit the dog’s gastrointestinal environment. Most of them belonged to Firmicutes , followed by Bacteroidota and Proteobacteria . We also assembled one Actinobacteriota and one Fusobacteriota MAG. CanMAGs harboured antimicrobial-resistance genes (ARGs) and prophages and were linked to plasmids. ARGs conferring resistance to tetracycline were most predominant within CanMAGs, followed by lincosamide and macrolide ones. At the functional level, carbohydrate transport and metabolism was the most variable within the CanMAGs, and mobilome function was abundant in some MAGs. Specifically, we assigned the mobilome functions and the associated mobile genetic elements to the bacterial host. The CanMAGs harboured 50 bacteriophages, providing novel bacterial-host information for eight viral clusters, and Hi-C proximity ligation data linked the six potential plasmids to their bacterial host. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAG discovery and assignment of extra-chromosomal elements to their bacterial host. This will provide essential information for studying the canine gut microbiome in veterinary medicine and animal nutrition.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 M... more We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 Mbp size and 99.43% completeness, 2,386 coding sequences, 19 complete rRNAs, 59 tRNAs, and 4 noncoding RNAs. We released 51 single-contig complete genomes and 16 genomes with a circular main contig using Nanopore sequencing. S taphylococcus pseudintermedius is a commensal of the skin of dogs and the main causative agent of canine pyoderma (1), but populations inhabiting the skin of healthy dogs are largely unknown. The current paradigm indicates that infection arises when the skin barrier is altered by predisposing factors (2). We previously demonstrated that Nanopore sequencing allows de novo assembly of the entire genome of S. pseudintermedius (3). Here, we retrieved 67 S. pseudintermedius genomes from isolates from the skin of healthy dogs using long-read Nanopore sequencing. Samples were obtained by rubbing sterile swabs on four skin sites from nine healthy dogs-perinasal, perioral, inguinal, and perianal. After culture in blood agar at 37°C for 24 h, colonies grown with the morphology of S. pseudintermedius (small silver colonies) were seeded in 3 mL of brain heart infusion (BHI) at 37°C for 16 h. DNA was extracted with a ZymoBIOMICS DNA miniprep kit (Zymo Research). DNA quality and quantity were determined using a NanoDrop 2000 instrument and a Qubit double-stranded DNA (dsDNA) broad range (BR) assay kit (Fisher Scientific). The sequencing libraries were prepared with the rapid barcoding sequencing kit (SQK-RBK004; Oxford Nanopore Technologies [ONT]). Up to 12 barcoded samples were loaded in a MinION FLO-MIN106 9.4.1 flow cell and sequenced in a MinION Mk1B or Mk1C device (ONT). The fast5 files were basecalled and demultiplexed, and adapters were trimmed with Guppy 5.0.11 (4) (ONT) (-dna_r9.4.1_450bps_sup.cfg) (-config configuration.cfg-barcode_kits SQK-RBK004-trim_barcodes; min_score threshold default 60). Reads with a quality score lower than 10 were discarded. Run summary statistics were obtained with Nanoplot 1.38.1 (5) (-N50-fastq). Isolates were confirmed as S. pseudintermedius by EPI2ME WIMP workflow (6). Genomes were de novo assembled using Flye 2.8.3 (7) (-nano-raw-plasmids-trestle), except HSP279 and HSP281, which were assembled with Flye 2.9 (-nano-hq). Contigs were polished with medaka 1.4.3 (8) (medaka_consensus;-m r941_min_sup_g507). Genome completeness and contamination were assessed with CheckM 1.1.3 (lineage_wf) (9). Circlator 1.5.5 was used to rotate the genomes, fixing the start with the dnaA gene (10) (fixstart-min_id 70). Genomes
Studies on tick microbial communities historically focused on tick-borne pathogens. However, ther... more Studies on tick microbial communities historically focused on tick-borne pathogens. However, there is an increasing interest in capturing relationships among non-pathogenic endosymbionts and exploring their relevance for tick biology. The present study included a total of 1600 adult ticks collected from domestic dogs in 4 different biogeographical regions of Spain. Each pool formed by 1 to 10 halves of individuals representing one specific ticks species was examined by PCR for the presence of Coxiellaceae, Rickettsia spp., Rickettsiales, Wolbachia spp., and other bacterial DNA. Of the pools analyzed, 92% tested positive for endosymbiont-derived DNA. Coxiella spp. endosymbionts were the most prevalent microorganisms, being always present in Rhipicephalus sanguineus sensu lato (s.l.) pools. Rickettsia spp. DNA was detected in 60% of Dermacentor reticulatus pools and 40% of R. sanguineus s.l. pools, with a higher diversity of Rickettsia species in R. sanguineus s.l. pools. Our study reveals a negative relationship of Rickettsia massiliae with the presence of tick-borne pathogens in the same pool of ticks. An additional endosymbiont, 'Candidatus Rickettsiella isopodorum', was only detected in D. reticulatus pools. Data from this study indicate that dogs in Spain are exposed to several endosymbionts. Due to the importance of tick-borne pathogens, characterizing the role of endosymbionts for tick physiology and prevalence, may lead to novel control strategies.
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